It is clinically more desirable to develop low density portable protein detection devices since the need for analyzing a large number of proteins in any one patient is less obvious. Such portable devices would enable clinicians to measure a few key proteins at the point of care such as small clinical laboratories and doctors' offices.
Unlike DNA assays, where high specificities can easily be predetermined by simple to chemical synthesis of the capture probe oligonucleotides and high sensitivity can be realized by employing a polymerase chain reaction (PCR) amplification step, the lack of a proteome-wide bioamplification strategy has been one of the practical bottlenecks to ultrasensitive protein assays. Furthermore, to build a viable protein array one has to choose a surface chemistry that will allow immobilized proteins or protein capturing bioaffinitive agents to retain their structural and biological integrities, and to devise an ultrasensitive and versatile means of measuring the degree of protein binding. Among the wide variety of approaches, sandwich immunoassays with optical detection are the most dominant in protein assays since they do not require the analyzed sample to be labelled. This greatly simplifies sample preparation and shortens assay time. The popularity of sandwich immunoassay advanced greatly in the 1970s with the introduction of enzyme-linked immunosorbant assay (ELISA) by Engvall and Perlman (Engvall, E.; Perlman, P. Immunochemistry 1974, 8, 871-874). It has become a standard technology in clinical laboratory.
Although optical detections have primarily been employed in ELISA, electrochemical transducers are more advantageous for such assays owing to their high sensitivity, inherent simplicity, and high portability. Furthermore, for opaque or optically dense matrices electrochemical methods are superior. For example, electrochemical measurements can be made on whole blood without interference from blood cells, other proteins, and fat globules. Electrochemical immunoassays (EIA) have therefore been studied as an attractive alternative to radioassays, fluorometric or colourometric assays. Of the many proposed EIAs, those employing amperometric detections have several distinct advantages over other EIAs, such as straightforwardness and high sensitivity. Traditionally, amperometric detection is carried in solution phase, relying on enzyme-mediated solution phase reaction for the formation of electroactive solution phase species. Due to dilution effect, detection limits were usually at ng/mL levels, which are inferior to ELISAs with the optical detections. To lower the detection limit of EIA, various amplification strategies, such as substrate-recycling, the use of nanoparticulate tags, adoption of DNA amplification techniques, including rolling circle amplification and biobarcode-based PCR, interdigitated array electrode, microfluidic devices, and magnetic beads have been proposed to improve the performance of EIA. For example, the biobarcode technology offers PCR-like sensitivity for proteins.
Nonetheless, new schemes, featuring improvements in simplicity, sensitivity or both and based on coupling the biocatalytic amplification of enzyme tags with additional to amplification units and processes are highly desired for meeting the high sensitivity demands of electrochemical detection of proteins.